JP2004069672A - Method and kit for measuring oxide appolipoprotein ai and oxide lipoprotein containing it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a kit simple and highly sensitive for measuring oxide appolipoprotein AI and/or HDL (oxide HDL), containing the oxide appolipoprotein AI. <P>SOLUTION: Tow kinds of antibodies, composed of the antibody which specifically react to an oxide part of the oxide appolipoprotein AI, and the antibody for the appolipoprotein AI or the antibody for a constitutional ingredient of the HDL are used in the method and kit specifically measure the oxide appolipoprotein AI and/or HDL (oxide HDL) containing the oxide appolipoprotein existing in a specimen by a sandwich method. By this invention, oxide stress in the living body can be detected. For an analysis of the mechanism or the clinical diagnosis of the oxide stress associated diseases, such as arteriosclerosis, complication disease of a diabetes mellitus (a nephropathy, a neuropathy etc.), the method and the kit can be utilized. <P>COPYRIGHT: (C)2004,JPO

Description

【００４８】
４−３：血清中酸化アポリポタンパク質ＡＩ
健常者４０名より得られた血清試料中の酸化アポリポタンパク質ＡＩを本ＥＬＩＳＡによって測定した。その結果、健常者の血中酸化アポリポタンパク質ＡＩは２９０．２２５±１６５．３ｎｇ／ｍｌ（ｍｅａｎ±ＳＤ）であった。
【００４９】
実施例２
＜材料と方法＞
ＩＩ型糖尿病患者から血液を採取し、得られた血清をサンプルとして使用した。なお、ＩＩ型糖尿病の診断は、ＷＨＯの基準　（Ｄｉａｂｅｔ　Ｍｅｄ．　１５：５３９−５３．（１９９８））　に従った。
血清サンプル中の酸化アポリポタンパク質ＡＩは、３Ｃ１１モノクローナル抗体を用いた上記ＥＬＩＳＡ法により測定した。統計解析はｓｔｕｄｅｎｔ−ｔ　ｔｅｓｔの片側検定を用い、Ｐ＜０．０５を有意水準とした。
【００５０】
＜結果＞
サンプルを採取した患者の臨床特徴を表２にまとめた。このようなサンプルを用い、血清中の酸化アポリポタンパク質ＡＩの濃度を測定したところ、図６に示すように、糖尿病性の腎症と神経障害の疾患陽性群において有意な酸化アポリポタンパク質ＡＩの濃度増加が認められた。
このことから、これら疾患の有病患者において酸化ストレスが亢進している可能性が示唆され、酸化アポリポタンパク質ＡＩが生体内の酸化ストレスマーカーならびに糖尿病合併症のマーカーとして利用可能であることを示唆した。
【００５１】
【表２】

【００５２】
【受託証】

【００５３】
【図面の簡単な説明】
【図１】図１は、固相された酸化及び未酸化のアポリポタンパク質に対する２種類の抗体反応性を示したものである。
【図２】図２は、固相された酸化及び未酸化のリポタンパク質に対する２種類の抗体反応性を示したものである。
【図３】図３は、ＥＬＩＳＡ法における標準曲線を示したものである。
【図４】図４は、ＥＬＩＳＡ法における交差性を示したものである。
【図５】図５は、血清サンプルの希釈性を示したものである。
【図６】図６は、糖尿病関連疾患から得られた血清サンプル中の酸化アポリポタンパク質Ａ１量を測定した結果を示したものである。[0001]
[Technology to which the Invention belongs]
The present invention relates to a method for immunologically measuring oxidized apolipoprotein AI and / or high-density lipoprotein (oxidized HDL) containing the antigen and a kit used therefor.
[0002]
[Prior art]
Previous epidemiological studies have shown that High Density Lipoprotein (HDL) has a prophylactic effect on arteriosclerosis. This prophylactic effect is believed to depend on the function of HDL to export cholesterol from the periphery. That is, primitive HDL produced by apolipoprotein AI receiving free cholesterol from the liver, the small intestine, etc., takes up cholesterol further by the LCAT enzyme while circulating to the periphery in the bloodstream to become mature HDL, and this mature HDL is obtained. Type HDL transports cholesterol accumulated in the periphery to the liver.
[0003]
Furthermore, it is becoming clear that the high oxidative sensitivity of HDL contributes to the anti-atherosclerotic effect. That is, HDL has a lower content of ubiquinol 10, which is an antioxidant, as compared with other lipoproteins, and is preferentially oxidized in vivo over LDL. For this reason, HDL is considered to suppress the production of oxidized LDL which is a causative substance of arteriosclerosis, and to be involved in the clearance of oxidized LDL in vivo.
[0004]
Such HDL is a lipoprotein having a high protein content of 50% as compared to other lipoproteins. The protein component of HDL is composed of apolipoproteins AI, AII, C and E, which contribute to the maintenance of HDL structure and the expression of functions. Among them, apolipoprotein AI accounts for about 70% of the protein component and is responsible for transporting cholesterol, which is a major function of HDL.
[0005]
When HDL is oxidized in a living body, it is known that apolipoprotein AI is also oxidized in parallel with oxidation of lipids, and oxidization of apolipoprotein AI causes aggregation and degradation of HDL. It is also known that the cholesterol transport ability of HDL decreases due to structural or chemical denaturation by oxidation.
[0006]
[Problems to be solved by the invention]
As described above, apolipoprotein AI greatly affects the anti-atherosclerotic effect of HDL and is very sensitive to oxidation. Therefore, oxidized apolipoprotein AI and / or HDL containing the antigen (oxidized HDL) is It is a marker for sensitive detection of oxidative stress in vivo, which has been strongly linked to the progress of lipid oxidation and arteriosclerosis, and the measurement of this marker is very useful for analysis of the mechanism of arteriosclerosis or clinical diagnosis. Conceivable.
[0007]
Conventionally, a method for measuring oxidized HDL using an antibody against oxidized lipid and an antibody against apolipoprotein AI (see JP-A-6-253888, JP-A-9-33525, and JP3251848) has been reported. However, a method for specifically measuring oxidized apolipoprotein AI and / or HDL containing the antigen (oxidized HDL) has not been reported.
[0008]
In the above-mentioned conventional method, apolipoprotein AI is mainly a protein component contained in HDL, and although an antibody against apolipoprotein AI is expected to have specificity for HDL, the lipid component of each lipoprotein is the same, Antibodies to the rendered lipids remain of concern for specificity for particular lipoproteins.
[0009]
Furthermore, it has been suggested that primitive HDL containing lipid-free apolipoprotein AI as a main component is dispersed in the interstitium and oxidized in arterial intima because of its small molecular size (Biochim. Biophys). Acta, 1483, 217-35 (2000)), specifically measuring oxidized apolipoprotein AI is HDL-specific and lipid-free primitive HDL and lipid-containing mature HDL It is expected that both HDL can be measured.
[0010]
[Patent Document 1]
JP-A-6-253888
[Patent Document 2]
JP-A-9-33525
[Patent Document 3]
Japanese Patent No. 3251848
[0011]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to develop a simple and highly sensitive method for measuring oxidized apolipoprotein AI. As a result, an antibody against apolipoprotein AI that specifically reacts with the oxidized site of oxidized apolipoprotein AI has been developed. Specific antibodies to oxidized apolipoprotein AI and / or high-density lipoproteins (oxidized HDL) containing oxidized apolipoprotein AI present in the sample by the sandwich method using two antibodies to We found that we could measure with sensitivity.
[0012]
As a result of examining the relationship between oxidized apolipoprotein AI and / or high-density lipoprotein (oxidized HDL) containing oxidized apolipoprotein AI and clinical symptoms using this measurement system, diabetic complications (nephropathy, neuropathy, etc.) ), Oxidized apolipoprotein AI is significantly increased, and oxidized apolipoprotein AI can be used not only as a marker for measuring the degree of oxidative stress in a living body but also as a diagnostic or predictive marker for diabetic complications. Was confirmed, and the present invention was completed.
[0013]
In Japan, one in four middle-aged and elderly people is thought to potentially have the potential to develop diabetes, which has a significant impact on patients' QOL and healthcare economy due to their complications. It is a disease that affects. There are many unclear points about the mechanism of the onset of diabetes and the complication of complications, and it has been pointed out that oxidative stress is involved in the onset thereof (Diabetes Metab Res Rev. 17: 189-212. (2001)). Diabetes Res. Clin. Pract. 45: 147-51 (1999), Am. J. Med. 30: 17S-26S (1999)), and reports on studies on prevention and prediction of diabetic complications using oxidative stress as an index. , Not found at the moment. Under such circumstances, it is quite unexpected that oxidized apolipoprotein AI can be used as a diagnostic or predictive marker for diabetic complications.
[0014]
Therefore, the present invention uses two types of antibodies, an antibody specifically reacting with an oxidation site of oxidized apolipoprotein AI as an antibody and an antibody against apolipoprotein AI, and oxidizes a sample present in a sample by a sandwich method. The present invention relates to a method and a kit for specifically measuring apolipoprotein AI.
[0015]
In addition, the present invention uses a sandwich method using two types of antibodies, an antibody specifically reacting with an oxidation site of oxidized apolipoprotein AI and an antibody against a component of high density lipoprotein (HDL). And a kit for specifically measuring high-density lipoprotein (oxidized HDL) containing oxidized apolipoprotein AI present in a sample.
[0016]
BEST MODE FOR CARRYING OUT THE INVENTION
The sample used in the present invention is not particularly limited as long as it contains oxidized apolipoprotein AI. Specifically, blood itself or a blood fraction such as serum or plasma can be used as the sample. Such a sample may be present in the form of oxidized apolipoprotein AI and / or HDL containing oxidized apolipoprotein AI (oxidized HDL). It is possible to measure even the form.
[0017]
The antibody used in the present invention depends on the antigen to be measured. For example, in the case of oxidized apolipoprotein AI and / or HDL containing oxidized apolipoprotein AI (oxidized HDL), the oxidized apolipoprotein AI And an apolipoprotein AI antibody, which specifically reacts with the oxidized site of the above, are used. When the antigen to be measured is HDL alone containing oxidized apolipoprotein AI (oxidized HDL), an antibody specifically reacting with the oxidized site of oxidized apolipoprotein AI and high-density lipoprotein (HDL) Two types of antibodies are used, one with the component (lipid or protein). Such an antibody may be an antibody itself or an active fragment thereof [F (ab ') ₂ , Fab ′, etc.].
[0018]
Here, the antibody specifically reacting with the oxidation site of the oxidized apolipoprotein AI is defined as an oxidation site of an amino acid constituting the oxidized apolipoprotein AI or a structural change of the apolipoprotein AI due to oxidation. Means an antibody that binds to In particular, in the present invention, a monoclonal antibody is preferably used as the antibody that specifically reacts with the oxidation site of the oxidized apolipoprotein AI, and a solid-phased antibody obtained by binding a monoclonal antibody having such specificity to a solid phase is preferred. By using it in a form, it is possible to measure oxidized apolipoprotein AI with higher sensitivity.
[0019]
An antibody that specifically reacts with the oxidized site of the oxidized apolipoprotein AI can be prepared by a conventional method. For example, using an oxidized apolipoprotein AI prepared using an oxygen radical generator or the like as an antigen, a monoclonal antibody is prepared by a conventional method, and it does not bind to unoxidized apolipoprotein AI from the obtained antibodies. An antibody that binds to the type of apolipoprotein AI may be selected.
[0020]
Further, an antibody against apolipoprotein AI is an antibody whose binding ability does not change even when apolipoprotein AI is oxidized and denatured. Such an antibody includes both apolipoprotein AI in HDL and free apolipoprotein AI. Recognize. Furthermore, an antibody against a component (lipid or protein) of high-density lipoprotein (HDL) is an antibody whose binding ability does not change even when HDL is subjected to oxidative denaturation. And / or recognize lipids.
Such an antibody may be either a monoclonal antibody or a polyclonal antibody.
[0021]
As the antibody against the apolipoprotein AI and the antibody against the HDL component, an antibody (polyclonal antibody, monoclonal antibody) can be prepared by a conventional method using the corresponding antigen (apolipoprotein AI, HDL or a component thereof). Further, such an antibody is already commercially available, and this commercially available product can be used.
[0022]
The present invention intends to measure oxidized apolipoprotein AI and / or HDL containing the antigen (oxidized HDL) in a sample by a sandwich method using the antibody as described above.
[0023]
The sandwich method itself does not change at all in the conventional method and procedure. However, as described above, the use of a solid-phased antibody in which a monoclonal antibody that specifically reacts with the oxidized site of oxidized apolipoprotein AI is bound to a solid phase as a solid-phase reagent enables oxidation to be performed with higher sensitivity. It is possible to measure the apolipoprotein AI and / or HDL (oxidized HDL) containing the antigen.
[0024]
As the carrier used when preparing the solid phase reagent, those usually used, for example, polyvinyl chloride, polystyrene, styrene-divinylbenzene copolymer, styrene-maleic anhydride copolymer, nylon, polyvinyl alcohol , Synthetic organic polymer compounds such as polyacrylamide, polyacrylonitrile, polypropylene, and polymethylene methacrylate; dextran derivatives (such as Sephadex); agarose gels (such as sepharose and biogel); and polysaccharides such as cellulose (paper disc and filter paper); Examples thereof include inorganic polymer compounds such as glass, silica gel, and silicone. These may be those into which functional groups such as amino group, carboxyl group, carbonyl group, hydroxyl group, and sulfhydryl group are introduced.
[0025]
The shape of the carrier can be flat (microtiter plate, disk, etc.), particulate (beads, etc.), tubular (test tubes, etc.), fibrous, membrane, fine particles (latex particles, etc.), capsule, vesicle The carrier may have any suitable shape depending on the measuring method.
[0026]
The binding between the carrier and the antibody can be performed by a known method such as a physical adsorption method, an ionic bonding method, a covalent bonding method, and an entrapment method [for example, “Immobilized enzyme” (edited by Ichiro Chibatake, March 20, 1975, Inc.) See Kodansha)), and the physical adsorption method is particularly preferred because it is simple. Further, the above-mentioned binding may be performed directly, or may be performed via another substance between both substances.
[0027]
The solid phase reagent thus obtained may be subjected to a blocking treatment using a usual blocking agent such as gelatin or BSA in order to suppress non-specific binding.
[0028]
As a labeling agent used for detection, a radioisotope (for example, ³² P, ³ H, ¹⁴ C, ¹²⁵ I), enzymes (eg, β-galactosidase, peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, catalase, glucose oxidase, lactate oxidase, alcohol oxidase, monoamine oxidase, etc.), coenzyme / prosthetic groups (eg, FAD) , FMN, ATP, biotin, heme, etc.), fluorescein derivatives (eg, fluorescein isothiocyanate, fluorescein thiol bamil, etc.), rhodamine derivatives (eg, tetramethylrhodamine B isothiocyanate, etc.), umbelliferone and 1-anilino-8 Fluorescent dyes such as -naphthalenesulfonic acid, luminol derivatives [for example, luminol, isoluminol, N- (6-aminohexyl) -N-ethyliso And the like can be exemplified Minoru like].
[0029]
The binding between the antibody and the labeling agent is described in a compendium (for example, “Seizoku Chemistry Laboratory Course 5 Immunobiochemical Research Method”, Tokyo Chemical Dojin, Ltd., (1986), pp. 102-112). What is necessary is just to implement by selecting suitably from such well-known methods.
[0030]
As a measurement procedure using such a solid phase reagent and an antibody reagent, a normal sandwich procedure may be employed as it is. That is, the solid phase reagent is reacted with the test sample and, if necessary, after BF separation, the antibody reagent is further reacted (two-step method), or the solid phase reagent, the test sample, and the antibody reagent are simultaneously reacted (one-step method). ), The oxidized apolipoprotein AI in the sample can be detected or quantified by a method known per se thereafter.
[0031]
For details of the sandwich method, refer to, for example, the following document.
(1) Hiro Irie edited by "Radio Immunoassay" (Kodansha Co., Ltd., issued May 1, 1979)
(2) Eiji Ishikawa et al., “Enzyme Immunoassay” (Second Edition) (Medical Publishing, December 15, 1982)
(3) Clinical Pathology Special Issue No. 53 “Immunoassay for Clinical Examinations-Technology and Application-” (Clinical Pathology Publishing Association, 1983)
(4) “Biotechnology Encyclopedia” (CMC Co., Ltd., issued on October 9, 1986)
5) “Methods in ENZYMOLOGY Vol. 70” (Immunochemical techniques (Part A))
(6) “Methods in ENZYMOLOGY Vol. 73” (Immunochemical techniques (Part B))
{Circle around (7)} “Methods in ENZYMOLOGY Vol. 74” (Immunochemical techniques (Part C))
(8) "Methods in ENZYMOLOGY Vol. 84" (Immunochemical techniques (Part D: Selected Immunoassay))
{Circle around (9)} “Methods in ENZYMOLOGY Vol. 92” (Immunochemical techniques) (Part E: Monoclonal Antibodies and General Immunoassay Methods)
[[5]-[9] are issued by Academic Press]
[0032]
The kit of the present invention is for carrying out the above-described assay method, and is composed of, for example, the following reagents, and a monoclonal antibody is preferred as the antioxidant apolipoprotein AI antibody.
(1) Immobilized antioxidant apolipoprotein AI antibody
(2) Labeled anti-apolipoprotein AI antibody or antibody against HDL component
[0033]
Further, a labeled anti-immunoglobulin antibody may be used in place of the labeled antibody, in which case it is composed of the following reagents.
(1) Immobilized antioxidant apolipoprotein AI antibody
(2) Anti-apolipoprotein AI antibody or antibody against HDL component
(3) Labeled anti-immunoglobulin antibody
In addition to the above reagents, if necessary, appropriately select an appropriate reagent according to the measurement method from each reagent such as a coloring reagent, a reaction stopping reagent, a standard antigen reagent, and a sample pretreatment reagent. Attach it.
[0034]
INDUSTRIAL APPLICABILITY The method and kit of the present invention can be used for detecting the degree of oxidative stress in vivo in animals including humans and detecting the oxidative stress-related disease. More specifically, as shown in Examples below, in a positive group such as diabetic complications (nephropathy, neuropathy, etc.), a significant increase in oxidized apolipoprotein AI is observed, By measuring oxidized apolipoprotein AI and / or HDL containing oxidized apolipoprotein AI (oxidized HDL), and comparing the measured value with the value of a healthy person or the reference value of the disease, arteriosclerosis, diabetic complications ( Nephropathy, neuropathy, etc.) can be detected.
[0035]
【The invention's effect】
According to the present invention, it has become possible for the first time to provide a simple and practical method for measuring oxidized apolipoprotein AI and / or HDL (oxidized HDL) containing oxidized apolipoprotein AI and a kit. Further, the method and the kit of the present invention have sufficient sensitivity for quantifying oxidized apolipoprotein AI and / or HDL containing oxidized apolipoprotein AI (oxidized HDL) in a sample, and detect oxidative stress in a living body. It can be used for analysis or clinical diagnosis of oxidative stress-related diseases such as arteriosclerosis and diabetic complications (nephropathy, neuropathy, etc.).
[0036]
【Example】
Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited thereto.
Example 1
<Method>
(1) Preparation of oxidized apolipoprotein
500 mg of human apolipoprotein AI, AII, B, CII, E (Chemicon, US) was used as an oxygen radical generator 2,2'-azobis (2-amidinopropane) dihydrochloride (4 mM) (AAPH; Wako Pure Chemical Industries, Ltd.) ) And reacted at 37 ° C. for 3 hours to obtain oxidized apolipoprotein.
[0037]
(2) Preparation of antioxidant apolipoprotein AI antibody
According to a conventional method, an antibody that strongly binds to oxidized apolipoprotein AI is produced from a hybridoma obtained by cell fusion of splenocytes of BALB / C mice immunized with oxidized apolipoprotein AI and myeloma cells SP2 / O-Ag14. Multiple clones were selected to obtain monoclonal antibodies. Among such antibodies, the monoclonal antibody 3C11 (FERM P-18759: deposited on March 12, 2002, National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary) was used.
[0038]
(3) Preparation of oxidized lipoprotein
Lipoprotein was purified from human EDTA plasma by density gradient specific gravity centrifugation. The obtained lipoprotein was further fractionated by gel filtration column chromatography (Biochem. J., 139, 89-95. (1974)), and only a fraction containing no lipoprotein other than the target lipoprotein was observed. Used for experiments. The protein content of the obtained lipoprotein was quantified by the method of Lowry et al., Adjusted to a concentration of 1 mg / ml, copper sulfate was added to these at 1 μM, and the mixture was incubated at 37 ° C. for 3 hours.
[0039]
(4) Antibody specificity test
The antigen specificity of antibody 3C11 against oxidized apolipoprotein AI and antibody against apolipoprotein AI (manufactured by Yamasa Corporation) were examined by immunological activity against each of the immobilized antigens. That is, each antigen (1 μg / ml: phosphate buffered saline (PBS), pH 7.4) was dispensed every 100 μl into a 96-well microplate (Nunc Immunoplate I), and reacted at 4 ° C. overnight. After removing the antibody solution by suction, the plate was washed with PBS, and each well was blocked with 300 μl of PBS containing 0.5% skim milk (all day and night at 4 ° C.). After removing the blocking solution by suction, 40 ng / ml antioxidant apolipoprotein AI antibody 3C11 diluted with PBS containing 1% bovine serum albumin and 1 ng / ml anti-apolipoprotein AI antibody (Yamasa) were reacted for 1 hour. After washing each well with PBS containing 0.05% Tween 20, 100 μl of an anti-mouse IgG antibody horseradish peroxidase-labeled product was added and reacted at room temperature for 1 hour.
After the reaction, the plate was washed with PBS containing 0.05% Tween 20, and a coloring solution (SAT-BLUE, Dojindo) was dispensed every 100 μl to develop a color. After stopping the reaction with 1N sulfuric acid, the absorbance was measured at 450 nm.
[0040]
(5) ELISA method
Monoclonal antioxidant rear polypoprotein AI antibody 3C11 solution was dispensed into a 96-well microplate (Nunc Immunoplate I) every 100 μl, and reacted at 4 ° C. overnight. After removing the antibody solution by suction, the antibody solution was washed with PBS, and blocked (4 ° C., overnight) with 300 μl of PBS containing 0.5% skim milk. After the reaction, the blocking solution was removed by suction, and the plate was dried. A sample or a standard concentration solution was added to each well of this 96-well microplate at 100 μl / well, and reacted at 37 ° C. for 3.5 hours. After washing with PBS containing 0.05% Tween 20, 100 μl of horseradish peroxidase-labeled anti-apolipoprotein AI antibody was added as a labeled secondary antibody, and the mixture was reacted at 37 ° C. for 1.5 hours.
After the reaction, the plate was washed with PBS containing 0.05% Tween 20, and a color developing solution (SAT-BLUE, Dojindo) was dispensed every 100 μl to develop a color. After stopping color development with 1N sulfuric acid, the absorbance was measured at 450 nm. The serum sample used was diluted 20-fold with 50 mM Tris-HCl (pH 7.4) containing 1% bovine serum albumin and 0.15 M sodium chloride. Apolipoprotein AI oxidized with 40 mM AAPH was used as a standard solution.
[0041]
<Result>
(1) Antigen specificity test
When the reactive specificity of the antioxidant apolipoprotein AI antibody 3C11 was tested using the antigen bound to the solid phase, it was found that the antibody showed specificity for oxidized apolipoprotein AI and oxidized HDL. (Figs. 1 and 2). This revealed that an antibody against oxidized apolipoprotein AI also reacted with oxidized apolipoprotein AI in HDL. More specifically, the absorbance when oxidized HDL is immobilized is 1.309, 6 times higher than that of unoxidized HDL, and the absorbance when oxidized apolipoprotein AI is immobilized is 1.009. And 4 times higher than that of unoxidized apolipoprotein AI. The absorbance of other apolipoproteins and lipoproteins other than HDL was 0.11 at the maximum, and no cross-reactivity was observed. On the other hand, the anti-apolipoprotein AI antibody showed no significant change in the absorbance due to the oxidation of HDL and apolipoprotein AI, and it was revealed that this antibody recognizes other than the part involved in the oxidation.
[0042]
(2) Standard curve
FIG. 3 shows a standard curve of the ELISA method prepared using a standard solution of oxidized apolipoprotein AI. The standard curve showed linearity from 2 ng / ml to 40 ng / ml, and good reactivity converging to the zero point was recognized.
[0043]
(3) Analysis of specificity of this ELISA method
Apolipoprotein AI and its oxide were measured by this ELISA method. From the measured results, the oxidized apolipoprotein AI concentration per 1 mg (ng / mg (protein)) was calculated, and the reactivity was compared (FIG. 4). As a result, apolipoprotein AI oxidized at 4 mM AAPH had a specificity 12 times higher than that of unoxidized apolipoprotein AI, and it was clear that this measurement system specifically recognizes oxidized apolipoprotein AI. became. Apolipoprotein AI oxidized at 40 mM AAPH was 730-fold higher than unoxidized apolipoprotein AI, and the specificity was further improved.
[0044]
When HDL and its oxide were measured by the present ELISA method, the oxidized HDL had a specificity of 8,500 times that of the unoxidized HDL (FIG. 4). The cross-reactivity to other apolipoproteins AII, CII, E and their oxides when 4 mM AAPH oxidized apolipoprotein AI was used as a control was 3% or less, and other lipoproteins (LDL, VLDL) and its oxides had a cross-reactivity of 0.05% or less (not shown).
[0045]
(4) Basic examination of this ELISA measurement method
4-1: dilution test of serum sample
A dilution linearity test in an oxidized apolipoprotein AI measurement system was performed using sera of healthy individuals showing a high value of oxidized apolipoprotein AI. As a result, all three tested samples showed good dilution linearity converging to the origin at a dilution factor of 10 or more (FIG. 5). Therefore, the subsequent measurement of the serum sample was performed at a 20-fold dilution.
[0046]
4-2: Addition recovery test
Oxidized apolipoprotein AI was added to the 10-fold diluted serum sample, and the recovery was observed. Table 1 shows the results. At all three concentrations tested, recovery was greater than 80%.
[0047]
[Table 1]

[0048]
4-3: Serum oxidized apolipoprotein AI
Oxidized apolipoprotein AI in serum samples obtained from 40 healthy persons was measured by this ELISA. As a result, the blood oxidized apolipoprotein AI of a healthy individual was 290.225 ± 165.3 ng / ml (mean ± SD).
[0049]
Example 2
<Materials and methods>
Blood was collected from a type II diabetic patient, and the obtained serum was used as a sample. The diagnosis of type II diabetes was in accordance with WHO criteria (Diabet Med. 15: 539-53. (1998)).
The oxidized apolipoprotein AI in the serum sample was measured by the above-mentioned ELISA method using a 3C11 monoclonal antibody. Statistical analysis used a one-tailed test of the student-t test, and P <0.05 was set as the significance level.
[0050]
<Result>
The clinical characteristics of the patients from whom the samples were taken are summarized in Table 2. Using such a sample, the concentration of oxidized apolipoprotein AI in the serum was measured. As shown in FIG. 6, a significant increase in the concentration of oxidized apolipoprotein AI was observed in the diabetic nephropathy and neuropathy disease positive groups. Was observed.
This suggests that oxidative stress may be increased in patients with these diseases, suggesting that oxidized apolipoprotein AI can be used as an in vivo oxidative stress marker and a marker for diabetic complications. .
[0051]
[Table 2]

[0052]
[Contract Certificate]

[0053]
[Brief description of the drawings]
FIG. 1 shows the reactivity of two antibodies to solid-phase oxidized and unoxidized apolipoprotein.
FIG. 2 shows the reactivity of two antibodies to oxidized and unoxidized lipoproteins immobilized.
FIG. 3 shows a standard curve in an ELISA method.
FIG. 4 shows crossing properties in an ELISA method.
FIG. 5 shows the dilutability of a serum sample.
FIG. 6 shows the results of measuring the amount of oxidized apolipoprotein A1 in a serum sample obtained from a diabetes-related disease.

Claims (8)

Using two types of antibodies, an antibody specifically reacting with the oxidized site of oxidized apolipoprotein AI and an antibody against apolipoprotein AI, the oxidized apolipoprotein AI present in the sample by the sandwich method is used. Method to measure specifically. The method according to claim 1, wherein the antibody specifically reacting with the oxidation site of the oxidized apolipoprotein AI is an immobilized antibody bound to a solid phase. It contains two types of antibodies, an antibody specifically reacting with an oxidation site of oxidized apolipoprotein AI and an antibody against apolipoprotein AI, and oxidizes apolipoprotein AI present in a sample by a sandwich method. Kit for specific measurement. The kit according to claim 3, wherein the antibody specifically reacting with the oxidation site of the oxidized apolipoprotein AI is an immobilized antibody bound to a solid phase. An antibody specifically reacting with an oxidation site of oxidized apolipoprotein AI and an antibody against a component of high-density lipoprotein (HDL) are used as antibodies, and are present in a sample by a sandwich method. A method for specifically measuring high-density lipoprotein (oxidized HDL) containing oxidized apolipoprotein AI. The assay method according to claim 5, wherein the antibody specifically reacting with the oxidation site of the oxidized apolipoprotein AI is an immobilized antibody bound to a solid phase. It contains two types of antibodies, an antibody that specifically reacts with an oxidation site of oxidized apolipoprotein AI and an antibody against a component of high-density lipoprotein (HDL), and is present in a sample by a sandwich method. A kit for specifically measuring high-density lipoprotein (oxidized HDL) containing oxidized apolipoprotein AI. The kit according to claim 7, wherein the antibody specifically reacting with the oxidation site of the oxidized apolipoprotein AI is an immobilized antibody bound to a solid phase.

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